Microbiome delivery platform

Abstract

The present invention relates to preservation of bioactive compositions and delivery of biological subjects. In some embodiments, the bioactive composition includes at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin, or mRNA. Some embodiments include vitrification of the bioactive composition, and preservation of the bioactive composition in a mixture of the vitrified intrinsic disorder protein and sugar. The vitrified bioactive ingredient is used to release the bioactive composition from the vitrified mixture. Some embodiments apply a vitrified bioactive composition to a biological subject such that a bioactive ingredient is applied to the microbiome of the biological subject. Some embodiments further provide for enteric encapsulation of the vitrified bioactive composition to protect the vitrified bioactive composition from the first set of environmental conditions but only released under the second set of environmental conditions.

Description

[0001] Priorities

[0002] This patent application claims priority to provisional U.S. patent application No. 63 / 236,857 filed on August 25, 2021, entitled “Microbiome Delivery Platform,” with Augustine Zvinavashe as inventor, the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] Embodiments of the present invention generally relate to protecting biologically active ingredients, and more particularly, various embodiments of the present invention relate to protecting biologically active ingredients and delivering the protected biologically active substances to a biological subject. Background Art

[0004] The practice of using herbs, leaves and some animal products to treat humans, plants and animals dates back to ancient times, but the trend of using fresh biological products in modern health and wellness is growing.

[0005] One challenge with using some biologics is maintaining the viability of the product during the process of converting the fresh material into a commercial product. In other words, the biological properties that make the product therapeutically useful are often destroyed during the process of converting it from a living state into a form that can be included in the product. Summary of the invention

[0006] One embodiment of the present invention provides a method for configuring a bioactive composition when delivering the bioactive composition to a biological subject. The bioactive composition includes at least one bioactive substance selected from microorganisms, cells, tissues, vaccines, probiotics, antibiotics, vitamins, peptides, RNAi or mRNA. The method also includes preserving the bioactive composition. The preservation method includes vitrifying the bioactive composition in a vitrified mixture composed of intrinsically disordered proteins and carbohydrates to produce a vitrified bioactive composition. The vitrified bioactive composition is used to release the bioactive composition from the vitrified mixture. The method also includes applying the vitrified bioactive composition to the biological subject, and the administration method includes applying the vitrified bioactive composition to the microbiome of the biological subject.

[0007] The method further includes providing an enteric package of the vitrified bioactive composition. The enteric package is used to preserve the vitrified bioactive composition under a first set of environmental conditions and can be decomposed under a second set of environmental conditions to release the vitrified bioactive composition.

[0008] Enteric packaging may include an enteric polymer capsule in which the vitrified bioactive composition is deposited, or may include an enteric coating that completely covers the vitrified bioactive composition.

[0009] The intrinsic disordered protein may be at least one of a tardigrade disordered protein, silk fibroin or an elastin fragment, and the sugar is a disaccharide. The silk fibroin may be extracted from silkworms. The disaccharide may be cellobiose, lactose or trehalose.

[0010] The microorganism may include at least one of the following: fungi, eubacteria, eukaryotes, or archaea.

[0011] The bioactive composition can be one of the following: anaerobic, aerobic, spore-forming, vegetative, native to the human microbiota, native to the animal microbiota, non-native to the human microbiota, non-native to the animal microbiota, an artificial version of a bioactive composition non-native to the human microbiota, or an artificial version of a bioactive composition non-native to the animal microbiota. The bioactive composition can be enhanced in activity by adding metabolites and / or preservatives.

[0012] The biological subject is a human. The administration method includes applying the vitrified bioactive composition to at least one part of the human, such as the skin, vagina, penis, foot or armpit. The administration method specifically includes ingesting the enteric-encapsulated vitrified bioactive composition in at least one form of an oral ingestible pill, an oral ingestible tablet, an ingestible tablet or a solution.

[0013] The vitrified bioactive composition is ingested in the form of an enteric-coated vitrified bioactive composition and enters the digestive tract of the biological subject. The enteric-coated vitrified bioactive composition can pass through the digestive tract intact until the enteric-coated vitrified bioactive composition encounters an environment with a pH value greater than about 6. When the pH value of the environment in which the enteric-coated vitrified bioactive composition is located is greater than about 6, it will decompose and release the vitrified bioactive composition. The vitrified bioactive composition can release the bioactive composition.

[0014] Vitrifying the bioactive composition can reduce the metabolism of the bioactive material in the bioactive composition, thereby causing the bioactive material to enter a quiescent state. Vitrifying the bioactive composition can protect the bioactive composition from death caused by oxygen exposure. The vitrified bioactive composition will not be released from the enteric-encapsulated vitrified bioactive composition in the stomach of a biological subject because the pH value of the stomach is less than about 5.

[0015] When the enteric-encapsulated vitrified bioactive composition encounters an environment with a pH value greater than about 6, the vitrified bioactive composition can be released from the enteric-encapsulated vitrified bioactive composition in the intestinal tract of the biological subject. The vitrified bioactive composition released in the intestinal tract can adhere to the intestinal mucosa, rehydrate, resuscitate and multiply. Over a period of time, the bioactive composition can be released from the vitrified bioactive composition. The bioactive composition released in the intestinal tract releases at least one active and healthy microorganism, cell, tissue, vaccine, probiotic, antibiotic, peptide, RNAi or mRNA. Prebiotics are included in the bioactive composition to provide energy to nourish the bioactive substances.

[0016] In some embodiments, the biological subject can be a plant, seed or soil. The administration method includes spraying the granules and / or powder solution containing the enteric-encapsulated vitrified bioactive composition on the plant, seed or soil.

[0017] Another embodiment of the present invention provides a method for making a composition of a biological subject microbiome include a bioactive composition. The bioactive composition includes at least one bioactive substance of a microorganism, cell, tissue, vaccine, probiotic, antibiotic, vitamin, peptide, RNAi or mRNA. The bioactive composition includes a vitrified mixture. The vitrified mixture includes intrinsically disordered proteins and sugars. The bioactive composition is preserved in the vitrified mixture to form a vitrified bioactive composition.

[0018] The composition also includes an enteric package. The enteric package is used to encapsulate the vitrified bioactive composition. The enteric package can store the vitrified bioactive composition under a first set of environmental conditions and release the vitrified bioactive composition under a second set of environmental conditions. Before being stored in the vitrified mixture, the bioactive material is freeze-dried. Further, the bioactive composition contains probiotics and / or antibiotics.

[0019] The vitrified bioactive composition can be used in skin care lotions. The vitrified bioactive composition can be used in wound healing medicines.

[0020] In some embodiments, the intrinsically disordered protein is a natural protein. In some embodiments, the intrinsically disordered protein is a synthetic polymer.

[0021] Another embodiment of the present invention may provide a method for configuring a bioactive composition when delivering the bioactive composition to a biological subject. The bioactive composition comprises a bioactive substance. The method comprises preserving the bioactive composition. The preservation method comprises vitrifying the bioactive composition in a vitrified mixture comprising intrinsically disordered proteins and sugars to produce a vitrified bioactive composition. The composition may release the bioactive composition from the vitrified mixture. The method comprises configuring an enteric package. The configuration method comprises encapsulating the vitrified bioactive composition in an enteric package. The method comprises applying the enteric-encapsulated vitrified bioactive composition to a biological subject. The administration method comprises applying the enteric-encapsulated vitrified bioactive composition to a microbial population of a biological subject.

[0022] In some embodiments, enteric encapsulation comprises encapsulating the vitrified bioactive composition in a capsule comprising an enteric polymer. In some embodiments, enteric encapsulation comprises coating the vitrified bioactive composition with an enteric material. In some embodiments, enteric encapsulation comprises coating a tablet formed of the vitrified bioactive composition with an enteric material.

[0023] The bioactive substance comprises at least one microorganism, cell, tissue, vaccine, probiotic, antibiotic, vitamin, peptide, RNAi or mRNA. The intrinsic disordered protein can be a tardigrade disordered protein, silk protein or elastin fragment, and the sugar is a disaccharide.

[0024] Another embodiment of the present invention provides a method for preserving a bioactive composition by preparing a vitrified solution. The preparation method includes providing a liquid, and adding an intrinsically disordered protein and a sugar to the liquid. The intrinsically disordered protein and the sugar are mixed in the liquid to form a vitrified solution. The bioactive composition is added to the vitrified solution and mixed, and then the liquid is removed from the bioactive composition in the vitrified solution. The liquid includes water or alcohol.

[0025] In some embodiments, the method further comprises heating the bioactive composition in the vitrification solution to a temperature between 20° C. and 125° C. In some embodiments, the method further comprises cooling the bioactive composition in the vitrification solution to a temperature between 0° C. and -80° C.

[0026] In some embodiments, removing the liquid comprises at least one of the following methods: air drying, freeze drying, vacuum drying, spray drying, or heat drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] For those skilled in the art, the advantages of various embodiments of the present invention should be more fully understood from the following overview drawings and "Description of Embodiments".

[0028] Figure 1AFIG. 1 is a schematic diagram of a process for preparing a vitrified bioactive composition according to an embodiment of the present invention.

[0029] FIG. 1B is a schematic diagram of a structure of a vitrified bioactive composition according to an embodiment of the present invention.

[0030] 1C is a schematic diagram of a process for preparing a vitrified bioactive composition for enteric encapsulation according to one embodiment of the present invention.

[0031] FIG. 2 is a schematic diagram of some potential applications of a vitrified bioactive composition according to an embodiment of the present invention.

[0032] FIG. 3 is a schematic diagram of a method for delivering a bioactive composition according to an embodiment of the present invention.

[0033] FIG. 4 is a schematic diagram of a method for delivering a bioactive composition according to an embodiment of the present invention.

[0034] FIG. 5A is a schematic diagram of a process for preserving a bioactive composition according to an embodiment of the present invention.

[0035] FIG. 5B is a schematic diagram of a process of preparing a vitrification solution according to an embodiment of the present invention. Specific embodiments

[0036] Some embodiments will be described in detail below to provide an overall understanding of the structure, function, manufacture and use principles of the disclosed systems, devices and methods. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the systems, components and methods specifically described herein and illustrated in the accompanying drawings are non-limiting embodiments, and the scope of protection of the present invention is defined only by the claims. The features described or illustrated in association with an embodiment may be combined with the features of other embodiments. Such modifications and variations should be included within the scope of protection of the present invention.

[0037] Active microbial cultures (e.g., natural or artificial) can improve the health of humans, animals, and plants by producing beneficial proteins and enzymes. A formulation containing proteins with intrinsically disordered regions and poly / di / monosaccharides [or proteins with intrinsically disordered regions only (synthetic or naturally occurring)] can be used to nourish, stabilize active microbial cultures (bacteria, protozoa, archaea, fungi in humans and animals), improve the performance of prebiotic and probiotic fragments, and control the release of the payload. The formulation can be used to maintain microbial viability in a liquid environment, freeze-dried state, and in dry powders or films.

[0038] Similarly, tardigrades can be kept alive in dry environments by extracellular vitrification using intrinsically disordered proteins and sugars, a method that preserves and protects microorganisms for long periods of time. This formulation may also provide additional benefits to the skin and intestines during delivery. The agents initially tested by the inventors included a solution of silk protein extracted from silkworms (a protein with disordered regions) and trehalose (a disaccharide). However, formulations of other intrinsically disordered proteins and disaccharides or monosaccharides (synthetic or naturally occurring) may also be used. Therefore, the discussion of these specific substances is for illustrative purposes only and is not intended to limit the various embodiments of the invention.

[0039] Artificial microorganisms, probiotics and metabolites can be encapsulated to maintain stability during manufacturing, storage and use. Such formulations are preferably used for human microbiota, including skin, vagina, underarms, or as pills, tablets or solutions for oral ingestion to nourish and protect the skin, and / or products for human and animal ingestion. In addition, prebiotics can be added to enhance the effects of microorganisms (bacteria, protozoa, archaea and fungi), and prebiotics and other substances will provide energy to nourish the microorganisms.

[0040] The embodiments disclose methods for preparing, preserving and delivering bioactive ingredients (e.g., bioactive substances) for treating animals and plants. The bioactive ingredients include at least one bioactive ingredient (e.g., a bioactive substance). The bioactive substance can be prepared, stored and delivered without killing (e.g., inactivating) the active ingredients of the bioactive substance so that the bioactive substance is delivered to the recipient with complete effectiveness. The bioactive substance and the bioactive composition can be preserved by vitrifying the bioactive substance and / or the bioactive composition in a vitrified mixture composed of intrinsic disordered proteins and carbohydrates to produce a vitrified bioactive composition. In some embodiments, the vitrified bioactive composition includes the above-described formulation. The vitrified bioactive composition is encapsulated in an enteric polymer package, which is used to protect the vitrified bioactive composition in some environments and release the vitrified bioactive composition in other environments. In an embodiment, the vitrified bioactive composition encapsulated in the enteric polymer package includes the formulation described above.

[0041] By vitrifying the bioactive composition and encapsulating the vitrified bioactive composition in an enteric polymer, the activity of the bioactive material and / or bioactive composition can be maintained and delivered to a biological subject under a predetermined set of environmental conditions. In some embodiments, the encapsulated vitrified bioactive composition (e.g., formulation) can be delivered to humans or animals via an orally swallowable pill, capsule, and / or solution. In some embodiments, the encapsulated vitrified bioactive composition can be delivered to plants, seeds, and / or soil by applying a powder and / or granules of the encapsulated vitrified bioactive composition, or by spraying a solution containing granules and / or powder of the enteric encapsulated vitrified bioactive composition on the plant, seed, or soil.

[0042] Bioactive substances

[0043] In some embodiments, bioactive materials (e.g., bioactive substances or bioactive ingredients) include microorganisms, cells, tissues, vaccines, probiotics, antibiotics, vitamins, and mRNA. Bioactive materials can interact with biological subjects (such as humans, animals, or plants).

[0044] In some embodiments, the bioactive material can be made into a bioactive composition containing multiple bioactive substances. The bioactive ingredients can also include metabolites and / or preservatives. Adding metabolites and / or preservatives can enhance the effectiveness of the bioactive composition.

[0045] In some embodiments, the bioactive material and / or bioactive composition may include fungi, eubacteria, eukaryotes, or archaea.

[0046] In some embodiments, the bioactive material and / or bioactive composition can be anaerobic, aerobic, spore-forming, vegetative, native to the human microbiota, native to the animal microbiota, non-native to the human microbiota, non-native to the animal microbiota, native to the plant microbiota, non-native to the plant microbiota, an artificial version of a bioactive composition non-native to the human microbiota, or an artificial version of a bioactive composition non-native to the animal microbiota.

[0047] In some embodiments, the bioactive substance and / or bioactive composition comprises one or more bacteria, metals, enzymes or biological products. For example, the metal comprises one or more alkali metals, alkaline earth metals or transition metals. In some embodiments, the biological product is insulin glajina, infliximab, rituximab, enalapril, adalimumab, monoclonal antibodies, trastuzumab or other biological products.

[0048] In some embodiments, the bioactive substance can be an oligonucleotide, such as RNA. The RNA can be tRNA, mRNA, rRNA, snRNA, srpRNA, gRNA, TERC, SL RNA, crRNA, miRNA, siRNA or eRNA.

[0049] In some embodiments, the bioactive substance can be an enzyme (i.e., RNase or DNase), a fatty acid, a sugar (e.g., alcohol sugar) or a mineral. For example, the enzyme can include pepsin, maltase, lactase, sucrase, disaccharidase, lipase, lysozyme, salivary amylase, pepsin, gastric lipase, other lipases, hydrochloric acid, intrinsic factor, mucin, gastrin, trypsinogen, cathelicidinase, elastase, etc.

[0050] In certain embodiments, additives may be added to the bioactive composition. The additive may be at least one colorant, chelating agent, ligand, antimicrobial agent, filler, spice or flavoring agent. For example, the colorant may be carmine, Ponceau 3R, purple carmine, Monascus, indigo, brilliant green SF, naphthalene yellow, orange 1, quinoline yellow, lemon yellow, E1 series (e.g., E100, E162, etc.), anthocyanin, betaine, carotenoid or phenols. In certain embodiments, the chelating agent may be ethylenediaminetetraacetic acid (EDTA), transferrin or deferiprone.

[0051] In some embodiments, the microorganism may be acetic acid, benzoic acid, natamycin, lysine, nitrate, nitrite, propionic acid, sorbic acid, sulfite, or sulfur dioxide. In other embodiments, the filler may be cellulose.

[0052] In some embodiments, the bioactive substance includes at least one vitamin, nutrient, antioxidant and protein. In some examples, the protein can be a peptide, an amino acid (e.g., a post-translationally modified amino acid) or a synthetic amino acid. The nutrient can be a mineral, protein, carbohydrate, fat, coenzyme Q10, glutathione, lithium, probiotics, glycine, DHA, flavonoids or others. Antioxidants include vitamins C and E, selenium, carotenoids, thiols, catalase, superoxide dismutase, uric acid and panthenol.

[0053] In other embodiments, the bioactive substances include at least one of green tea extract, rosemary extract, phenolic antioxidants, catechins, acerola, tocopherol, chamomile extract, arnica, tea tree (Camellia sinensis), epicatechin, epigallocatechin, epigallocatechin gallate, vitamin A, vitamin E and / or vitamin C.

[0054] In some embodiments, the additive is mixed with an accelerator or an excipient. For example, the additive is mixed with polyethylene glycol or xylitol. Furthermore, the additive can be emulsified with an accelerator or an excipient and mixed into the silk fibroin solution.

[0055] Vitrification

[0056] In some embodiments, the bioactive substance and / or bioactive composition will be mixed with a vitrified combination of structural proteins and sugars. Structural proteins and sugars can be mixed with a solution to prepare a vitrified solution. Vitrification is the process of converting a substance into an amorphous (e.g., glass) substance. Under predetermined conditions, intrinsic disordered proteins and sugars will undergo vitrification together with the bioactive substance and / or bioactive composition.

[0057] For example, under a pressure lower than atmospheric pressure, the slow annealing process includes forming a vitrified solution from a combination of a bioactive substance and / or a bioactive composition and an intrinsic disordered protein and a sugar at room temperature and pressure in a reaction vessel. The reaction vessel is slowly evacuated at room temperature for 2-12 hours to produce an amorphous material having a highly ordered local layered structure of silk protein and disaccharides on its surface, which is referred to as a "vitrified bioactive composition". In some other aspects of the embodiments, the silk fibroin solution can be dehydrated by air drying, freeze drying, vacuum drying, spray drying or heat drying.

[0058] The density of this highly ordered layered material is high enough to significantly slow the rate of exchange of oxygen from the surrounding air to the vitrified bioactive composition and vice versa. This vitrification process effectively suspends the activity of the bioactive material and / or bioactive composition (e.g., reduces its metabolism and puts it into a quiescent state) without killing it. In addition, the vitrified state protects anaerobic bioactive materials and / or anaerobic bioactive compositions (e.g., materials that cannot survive in the presence of oxygen) from death due to exposure to oxygen under normal temperature and pressure conditions.

[0059] When vitrification is performed on anaerobic bioactive materials and / or bioactive compositions, the entire annealing process must be performed in an anaerobic reaction vessel under anaerobic conditions. Only after the mixture is completely annealed into a vitrified bioactive composition can the anaerobic microorganisms be exposed to oxygen (e.g., air). Generally speaking, the materials used in the vitrification process are safe and can therefore be ingested by humans.

[0060] Intrinsically disordered proteins

[0061] As described above, the vitrified mixture includes an intrinsically disordered protein and a carbohydrate. An intrinsically disordered protein is a structural protein that can be a naturally occurring protein or a synthetic polymer. An intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure, typically in the absence of its macromolecular interaction partners.

[0062] In some embodiments, structural proteins (e.g., intrinsically disordered proteins) are proteins composed of repeating units that form an ordered structure (e.g., a β-sheet), while non-repeating regions of the amino acid chains form disordered secondary and tertiary structures. In some embodiments, the structural protein is selected from fibroin, suture protein, or keratin.

[0063] In some embodiments, the structural protein is silk fibroin. In some embodiments, the silk fibroin is extracted from silkworm cocoons. Silk fibroin is a structural protein best known for its use in textiles and has been reengineered as a naturally derived technological material for applications in regenerative medicine, drug delivery, implantable optoelectronics, and food coatings.

[0064] Silk fibroin is a structural protein that can be produced and extracted from silkworms, spiders, or other insects, or it can be generated synthetically. Natural silk fibroin is produced from species such as Antheraea pernyi, Giant Lichen Web Spider, Araneae, Bombyx mori, Bombyx mori, Galleria mellonella, Nephila scutellaria, Madagascar Golden Silk Orb-weaver, and Araneae scutellaria. The unique properties of silk fibroin derive from its structure, which consists of hydrophobic blocks separated by hydrophilic spacers. In its natural state, silk fibroin is organized in a β-sheet formation, consisting of alternating highly ordered crystalline and amorphous regions. This unique structure gives silk fibroin-based materials high strength and toughness. Silk fibroin solutions can be processed into a variety of morphologies, making them attractive for a number of high-tech applications, including tissue engineering scaffolds, bone screws for fixation, and drug depots for therapeutic delivery.

[0065] Among other insects, the silkworm begins its life cycle in a cocoon. A single cocoon can consist of hundreds of meters of silk. The cocoon itself is primarily composed of two proteins: fibroin and sericin. Along with other components, fibroin helps regulate the diffusion of gases in and out of the cocoon. Similarly, this regulation can also be achieved with fibroin-based materials. This has applications in the preservation of bioactive materials, as the main deterioration modes are associated with oxidation, degradation, and dehydration processes. When applied to bioactive ingredients, the tunability of silk fibroin allows for diverse applications across different types of bioactives. Silk fibroin forms polymer coatings with different solubility and gas permeability. This is finely tuned by different production parameters such as concentration, shear force, additives, temperature, and post-treatment. By adjusting these parameters, the structural organization of silk fibroin can be controlled. The structural differences ultimately affect crystallinity, elasticity, solubility, and porosity, which in turn change the diffusion behavior of gases and water, as well as reduce the growth rate of microorganisms. Silk fibroin coatings can be designed for different storage conditions or products of interest.

[0066] The structural protein, silk fibroin, is purified from silkworm cocoons through a water-based process that uses hybrid ions like LiBr to break the inter- and intramolecular hydrogen bonds that cross-link the silk fibroin molecules into fibers. After ions are removed by dialysis, the silk fibroin exists as nano-micelles in aqueous suspension, a state that is stable for a period of time ranging from days to months, depending on concentration, pH, and molecular weight.

[0067] The assembly of materials is driven by the dehydration process and the formation of new intra- and intermolecular hydrogen bonds. A variety of material forms can be obtained through this process, including transparent, tough films, which have been applied to extend the shelf life of perishable cooked foods. Silk fibroin, with a diblock copolymer-like structure, has hydrophobic repeating amino acid sequences separated by hydrophilic, negatively charged non-repeating sequences, making silk fibroin polymorphic as the protein can be obtained as a random coil or a β-sheet-rich structure, making the silk material water-soluble or water-insoluble, respectively. The silk fibroin structure also provides a unique environment that can protect labile compounds ranging from antibiotics to growth factors, enzymes, and viruses by alleviating oxidative stress, providing sufficient hydration, and maintaining the conformation of biomolecules under anhydrous conditions.

[0068] Silk fibroin extracted from the silkworm (e.g., Bombyx mori) is a natural protein. Another natural protein includes silk fibroin extracted from tardigrades. Tardigrades are tiny aquatic animals that can exhibit amazing resistance to environmental stresses such as extreme temperatures, radiation, high pressure, or lack of oxygen or water. Faced with such stresses, tardigrades transform into a special desiccation state that allows them to survive for long periods of time. Although the molecular mechanisms that confer protection from such extreme physiological stresses are still unclear, studies have revealed the importance of two families of intrinsically disordered proteins unique to tardigrades: cytoplasmic abundant heat-soluble (CAHS) proteins and secretory abundant heat-soluble (SAHS) proteins. These tardigrade-specific intrinsically disordered proteins include cytoplasmic abundant heat-soluble (CAHS) proteins and secretory abundant heat-soluble (SAHS) proteins. These proteins are collectively referred to as tardigrade disordered proteins (TDPs). RNA interference experiments have shown that TDPs are essential for desiccation tolerance in tardigrades, and heterologous expression of some TDPs has demonstrated that they can increase desiccation tolerance in yeast and Escherichia coli.

[0069] In some embodiments, elastin can be used as an intrinsic disordered protein (e.g., IDPs) for combination. Elastin exists in the extracellular matrix as a fiber and / or discontinuous sheet structure, giving it the characteristics of stretching and elastic rebound. Elastin is a protein synthesized by fibroblasts and exists in a monomer precursor form called preelastin. In the extracellular matrix, monomers are polymerized by the enzyme lysine oxidase, and the lysine amino acid side chains are extensively cross-linked. The deposition of elastin in the form of fibers requires the presence of microfibril templates of structural glycoprotein fibrin and related glycoproteins. These are integrated around and inside the final elastic fibers. Preelastin is a soluble precursor of elastin, secreted in the form of a monomer of 60-70kDa by cells that produce elastin, such as fibroblasts, alveolar cells, chondrocytes, and vascular smooth muscle cells. After secretion, preelastin has a tendency to self-aggregate, a process known as coagulation. The arrangement of preelastin aggregates on the cell surface is mediated by interaction with cell surface proteoglycans.

[0070] Elastin is both the name of the fiber and the name of the polymerized protein. There are also two related fibers, oxidized fibers and serine fibers, which contain more fibrous protein and less prepolymerized elastin than ordinary elastin. In some embodiments, preelastin is used as an intrinsic disordered protein (IDP) together with sugar to form a vitrified mixture.

[0071] In some embodiments, synthetic polymers can be used in combination as intrinsically disordered proteins (e.g., IDPs). For example, elastin-like polypeptides (e.g., ELPs) are a class of laboratory-designed intrinsically disordered proteins that, although not usually found in nature, share many of the same properties as natural intrinsically disordered proteins, including temperature responsiveness and salt / pH sensitivity. ELPs can be used as a set of genetically encoded amino acids. They can be fused with various ribonucleic acid (e.g., RNA) binding proteins and form synthetic IDP-RNA enriched particles. These synthetic particles can bind to deoxyribonucleic acid (DNA) and RNA. In some embodiments, synthetic IDP-RNA particles can be used to replicate natural IDP-RNA particles to achieve the delivery of DNA and RNA into biological systems as a disease treatment method. ELPs can be fused with targeting proteins for specific mRNAs to regulate gene expression by isolating targeted mRNAs using the temperature-responsive phase separation activity of ELPs.

[0072] Synthetic polymers can be used as intrinsically disordered proteins (IDPs) for combination to improve specific properties of vitrified bioactive compositions. For example, some synthetic IDPs can have higher temperature tolerance. Other synthetic IDPs can have higher radiation tolerance.

[0073] sugar

[0074] As described above, the sugar can be combined with the intrinsic disordered protein to form a glassy mixture. In some embodiments, the sugar is a disaccharide. The disaccharide can be selected from trehalose, sucrose, lactose or maltose. In some embodiments, the disaccharide is trehalose.

[0075] Trehalose is a non-reducing disaccharide in which two glucose units are linked by an α,α-(1,1)-glycosidic bond. This disaccharide can be isolated from all living organisms, including plants, animals, fungi, yeasts, archaea and bacteria. Trehalose is also produced industrially because it plays a role in the food, cosmetics and pharmaceutical industries. This disaccharide can play important and specific roles in cells, such as as a signaling molecule, carbohydrate reserve and protector against various stressful environments such as drought, cold and salt. The accumulation of trehalose occurs both inside and outside the cells.

[0076] Forming a vitrified bioactive composition

[0077] As described above, in some embodiments, a bioactive composition (e.g., formulation) is combined with a mixture of intrinsically disordered proteins (e.g., IDPs), such as silk fibroin, and sugars, such as disaccharides, to produce a vitrified bioactive composition. The combination of IDPs and sugars can be mixed with a solvent to prepare a vitrified solution. The vitrification process provides a microenvironment for the bioactive component (e.g., bioactive substance) bioactive composition. Useful solvents include water and alcohols.

[0078] The detailed formation mechanism of sugar-driven cell protection includes that sugars (e.g., trehalose) form a glassy matrix inside cells, which physically prevents protein denaturation, protein aggregation, and membrane fusion of IDPs. In IDPs and sugar solutions, hydrogen bonds between water and cellular components are replaced by trehalose when cells dry, which can also prevent protein denaturation of IDPs and aggregation and membrane fusion of sugars. That is, in the presence of trehalose, hydrogen bond replacement between silk fibroin molecules and between water molecules is thermodynamically favorable, and despite trehalose-induced protein vitrification, the protein can still undergo structural reorganization, water loss, and volume change.

[0079] In some embodiments, the vitrified bioactive composition comprises an intrinsically disordered protein (IDP) to sugar ratio (e.g., IDP:sugar) ranging from about 10:1 to about 1:10. In some embodiments, the ratio of IDP to sugar in the vitrified bioactive composition is from about 5:1 to about 1:5. In some embodiments, the ratio of IDP to sugar in the vitrified bioactive composition is from about 3:1 to about 1:3. In some embodiments, the ratio of IDP to sugar in the vitrified bioactive composition is about 1:1. In some embodiments, the vitrified bioactive composition comprises a mixture of multiple IDPs and sugars.

[0080] Preservation of biomolecules in silk fibroin formulations is associated with matrix β-relaxation, just as in sugar-based dry formulations. Inclusion of sugars like sucrose in silk fibroin-based materials enhances protein stabilization, as they act as antiplasticizers to inhibit β-relaxation and slow degradation rates. Trehalose does not interfere with silk fibroin assembly, as its addition to silk fibroin suspensions does not cause any changes to the random coiled structure presented by silk fibroin, nor does it drive protein assembly.

[0081] In some embodiments, additives may be added to the vitrified solution. These additives may include at least one sugar, plasticizer, or crosslinking agent. For example, the sugar additive may be a sugar alcohol, a polyol, or a hygroscopic polymer (e.g., polyethylene glycol). In some embodiments, the sugar additive is a crosslinking agent that may be photosensitive.

[0082] Preparation of bioactive compositions of IDPs and sugars

[0083] The preparation process of combining the bioactive ingredients with a vitrification solution (eg, a mixture of IDPs and sugars in a solvent) to form a vitrified bioactive composition to preserve and protect the bioactive composition from damage can be achieved by a variety of methods.

[0084] In some embodiments, the vitrification solution can be deposited onto the bioactive composition by spray coating. Alternatively, the vitrification solution can be deposited onto the bioactive composition by dip coating.

[0085] In some aspects, the vitrified solution may not need to be annealed before or after deposition. In some embodiments, the bioactive composition may include multiple layers of silk fibroin / sugar deposited from a vitrified solution. For example, the bioactive material may be sprayed with a vitrified solution, dried, and then sprayed again. This process may be repeated any number of times to increase thickness and additional layers. Further, in some embodiments, the bioactive composition may include multiple layers, each with a specific function. For example, the bioactive material may be coated with silk fibroin / sugar. Then, the silk fibroin / sugar-coated bioactive material itself may be wrapped with another layer of coating that is hydrophobic or waterproof, so that water cannot penetrate the outer layer and reach the inner silk fibroin / sugar layer.

[0086] Further, in some embodiments, tablet coating can be used, wherein the silk fibroin / sugar layer is coated in an industrial drum. In addition, tablet coating and film coating can also be used.

[0087] In some embodiments, the preservation of the bioactive composition is for a bioactive composition comprising silk fibroin and a sugar, wherein the silk fibroin is previously in powder form, or in a solution of powdered silk fibroin reconstituted in a solvent.

[0088] In some embodiments, the bioactive composition is stored in a solution containing silk fibroin and a sugar. In some embodiments, the solution may contain at most 25% of the silk fibroin fragments. Further, in some embodiments, at most only 10% of the silk fibroin fragments have a molecular weight exceeding 400 kilodaltons (kDa). Further, in some other aspects, at most only 45% of the silk fibroin fragments have a molecular weight exceeding 300 kDa.

[0089] In some embodiments, the preservation of the bioactive composition is for a bioactive composition comprising a matrix, such as a powder, a pill, a tablet, a capsule, etc. The matrix can be covered with at least one layer of silk fibroin / sugar layer. In some embodiments, the thickness of at least one layer of silk fibroin / sugar layer can vary from about 12 nanometers (nm) to about 180 micrometers (μm).

[0090] In some embodiments, preservation is directed to a method for preparing a bioactive composition. In some embodiments, the bioactive composition may be sprayed with a solution containing silk fibroin fragments. In some embodiments, the molecular weight of the silk fibroin fragments is between 10 kilodaltons (kDa) and 600 kilodaltons (kDa).

[0091] In some embodiments, the preservation of the bioactive composition is directed to a method for preparing the bioactive composition. In some embodiments, the bioactive composition may include dip-coating the bioactive composition with a solution containing silk fibroin fragments. In some embodiments, the molecular weight of the silk fibroin fragments is between 10 kilodaltons (kDa) and 600 kilodaltons (kDa).

[0092] In some embodiments, preservation is directed to a method for preparing a bioactive composition. In some embodiments, the bioactive composition can include mixing the bioactive composition with silk fibroin fragments. In some embodiments, the molecular weight of the silk fibroin fragments is between 10 kilodaltons (kDa) and 600 kilodaltons (kDa).

[0093] In some embodiments, the preservation of the bioactive composition is directed to a method of preparing the bioactive composition. In some embodiments, the bioactive composition is mixed with or covered with silk fibroin only. That is, in some embodiments, the vitrified bioactive composition does not include sugar.

[0094] In some embodiments, the preservation of the bioactive composition is directed to a method of preparing the bioactive composition. In some embodiments, the bioactive composition is only mixed with or covered with sugar. That is, in some embodiments, the vitrified bioactive composition does not include silk fibroin.

[0095] Enteric polymers

[0096] An enteric polymer is a polymer barrier applied to a bioactive ingredient that prevents it from dissolving or decomposing in certain environments while allowing it to dissolve or decompose in other environments. A common example is the application of a polymer barrier to an oral drug to prevent it from dissolving or decomposing in the gastric environment. That is, an enteric coating prevents a polymer-coated oral drug from dissolving or decomposing in the stomach, allowing the drug to be delivered to the lower gastrointestinal tract. Some drugs are unstable at the pH of gastric acid (about 2) and need to be protected from degradation. Enteric coatings are also an effective way to achieve drug targeting (such as gastric acid resistant drugs). Other drugs, such as some anthelmintics, may need to achieve higher concentrations in specific parts of the intestine. Enteric coatings can also be used as a research tool in studies to determine drug absorption. Enteric-coated drugs fall into the category of "delayed action" dosage forms.

[0097] Tablets, tablets, pellets and granules (usually enclosed in a capsule shell) are the most common enteric coated dosage forms. Enteric polymers can be applied to the surface of tablets, tablets, pellets and powders. Each tablet, tablet, pellet and powder is covered with an enteric polymer coating so that each particle in the powder, pellet, tablet and tablet is completely covered. That is, no matter how large the specific bioactive ingredient is, the specific coating is an enteric polymer coating.

[0098] Enteric polymers can also be used as a coating for capsules. In such embodiments, the vitrified bioactive composition is encapsulated in a capsule having an enteric polymer coating. In some embodiments, the vitrified bioactive composition is encapsulated in a capsule made entirely of an enteric polymer.

[0099] Enteric coatings protect the gastric mucosa from the irritation of the drug itself by preventing the bioactive composition from dissolving in the stomach. When the drug reaches the neutral or alkaline environment of the intestine, its active ingredients can dissolve and be absorbed into the blood. Bioactive compositions that are irritating to the stomach can be coated with a substance that only dissolves in the small intestine. Similarly, some bioactive compositions can be acid-activated. For such bioactive compositions, the enteric coating in the formulation acts more to prevent the drug from being activated in the mouth and esophagus.

[0100] Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. The material traditionally used is a resin film solution.

[0101] Microbiome

[0102] The microbiome is defined as a characteristic community of microorganisms occupying relatively well-defined activity areas with distinctive physicochemical properties. The microbiome refers not only to the microorganisms involved but also to their areas of activity, which leads to the formation of specific ecological niches. The microbiome is a dynamic, interacting microecological system that is prone to change over time and scale and is an integral part of the macroecological system, including the eukaryotic host, which is essential for their function and health.

[0103] The microbiota, although distinct from the microbiome, is interconnected and consists of microorganisms belonging to different kingdoms, including prokaryotes (bacteria, archaea) and eukaryotes (algae, protozoa, fungi, etc.), while “their domain of activity” includes microbial structures, metabolites, mobile genetic elements (such as transposons, phages, and viruses), and remnants of genetic material embedded in the environmental conditions of the habitat.

[0104] In some embodiments, a bioactive substance and / or bioactive composition is delivered to a microbiome. The bioactive substance / bioactive composition may contain a microbial population specific to the microbiome to which it is delivered.

[0105] Delivery form

[0106] By vitrifying the bioactive composition and encapsulating the vitrified bioactive composition in an enteric polymer, the bioactive material and / or bioactive composition can be preserved in vivo and then delivered to the microbiome of a biological subject under a predetermined set of environmental conditions.

[0107] Some embodiments describe methods for protecting, preserving and controlling the release of microorganisms and other bioactive ingredients to various microbial communities such as the skin, mouth, vagina and intestines of humans and animals. In some embodiments, it can also be used in other parts of the body. In other states, some embodiments may be in the form of liquid, freeze-dried, gel or film formulations (e.g., vitrified bioactive compositions), which may be composed of intrinsically disordered proteins and disaccharides and prebiotics that can artificially simulate the environment of microorganisms and other bioactive ingredients to ensure that it can obtain nutrition and is protected from dehydration, temperature, oxygen and water vapor pressure. Some embodiments describe methods for protecting, preserving and controlling the release of microorganisms and other bioactive ingredients to various microbial communities such as plants, seeds and soil.

[0108] In some embodiments, the encapsulated vitrified bioactive composition (e.g., formulation) can be delivered to humans or animals via orally ingestible pills, capsules, powders, granules, and / or solutions. The bioactive composition released in the intestinal tract of the biological subject releases at least one active and healthy microorganism, cell, tissue, vaccine, probiotic, antibiotic, peptide, RNAi, or mRNA.

[0109] In some embodiments, the encapsulated vitrified bioactive composition (e.g., formulation) can be delivered to humans or animals via orally ingested pills, capsules, powders, pellets, and / or solutions. The bioactive composition released in the intestinal tract of the biological subject releases at least one of live and healthy microorganisms, cells, tissues, vaccines, probiotics, antibiotics, peptides, RNAi, or mRNA.

[0110] In some embodiments, the encapsulated vitrified bioactive composition can be delivered to plants, plant seeds, and / or soil by applying a solution, powder, and / or pellets of the encapsulated vitrified bioactive composition. The encapsulated vitrified bioactive composition can be delivered to plants, plant seeds, and / or soil by spraying a solution containing particles and / or powder of the enteric encapsulated vitrified bioactive composition onto the plants, seeds, or soil.

[0111] In some embodiments, the vitrified bioactive composition can be delivered to humans, animals, and / or plants without being encapsulated by an enteric polymer.

[0112] In some embodiments, the vitrified bioactive composition can be delivered to humans, animals, and / or plants via enteric polymer encapsulation.

[0113] In other states, some embodiments of the bioactive composition can be in the form of a liquid, freeze-dried, gel or film-like preparation, and the preparation can be composed of intrinsically disordered proteins and disaccharides and / or prebiotics that can modify the environment of the microorganism (e.g., biological activity) to ensure that it has nutrients and is protected from desiccation, temperature, oxygen and water vapor pressure.

[0114] In embodiments, the vitrified bioactive composition may contain plant microorganisms and other plant bioactive substances as biofertilizers to replace the plant growth benefits of previous herbicides, pesticides and synthetic fertilizers. The use of silk fibroin / sugar dry films as seed coatings can be used to locate and quantify the delivery of plant microorganisms, thereby alleviating the effects of plant stress and soil salinity. Many different types of microorganisms and other plant bioactive substances can be delivered using the same technology.

[0115] In some embodiments, the bioactive composition can be implemented as a biodegradable material with intrinsic disordered proteins (IDPs) and disaccharides, which will be used to preserve symbiotic intestinal microorganisms at room temperature or near room temperature under aerobic conditions. IDP is any protein that is substantially lacking in secondary structure in aqueous solution, such as silk fibroin from silkworm. The encapsulated microorganisms can be fungi, true bacteria, protists or archaea in nature. They can be anaerobic or aerobic (referring to oxygen-intolerant or oxygen-tolerant, respectively), spore-forming or trophic, native to human or animal microbial groups, non-native but proven to be beneficial to human or animal microbial groups, or artificial versions of the first two types of microorganisms. The effect of the mixture can be enhanced by adding some metabolites or preservatives.

[0116] Figure 1A 1 is a schematic diagram of a method for preparing a vitrified bioactive composition comprising one or more bioactive ingredients according to an embodiment. The method 100 preserves the bioactive composition by maintaining the activity of the one or more bioactive ingredients and preventing the bioactive ingredients from deteriorating due to oxidation, degradation and / or dehydration.

[0117] Briefly, bioactive composition 110, intrinsically disordered proteins (IDPs) 120, and sugar 130 are added to and mixed in liquid 115 to form the bioactive composition in vitrified mixture solution 145. Liquid 115 is then removed to form a vitrified bioactive composition retained in container 170.

[0118] like Figure 1A As shown, a bioactive composition 110 is added to a liquid 115. The bioactive composition 110 may include at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin, or an mRNA.

[0119] In addition, intrinsically disordered proteins (IDPs) 120 are also included in the bioactive composition 110 in the liquid 115. IDPs are structural proteins that lack a fixed or ordered three-dimensional structure, and are generally lacking their macromolecular interaction partners. Silk fibroin is a non-limiting example of a structural protein that can be produced and extracted from silkworms, spiders, or other insects. It can also be produced synthetically by other methods.

[0120] Silk fibroin is a structural protein best known for its use in textiles that has been reinvented as a naturally derived technological material for applications in regenerative medicine, drug delivery, implantable optoelectronics, and food coatings. The unique properties of silk fibroin derive from its structure, which consists of hydrophobic blocks separated by hydrophilic spacers. In its natural state, silk fibroin is organized in a β-sheet formation consisting of highly ordered crystalline regions alternating with amorphous regions. Amorphous and crystalline regions are identified in the schematic diagram of IDPs, as shown in Figure 2. Figure 1A .

[0121] In addition, if Figure 1A As shown, sugar 130 is added to intrinsically disordered proteins (IDPs) 120 and bioactive composition 110 in liquid 115. Sugar 130 is used for illustrative purposes and is non-limiting because sugars are generally useful for making glassy mixtures. For example, the sugar can be in the form of a sugar, a disaccharide, a monosaccharide, and / or a monosaccharide.

[0122] As mentioned above, Figure 1A is a schematic diagram of bioactive composition 110 , IDPs 120 , and sugar 130 added to 140 and mixed in liquid 115 to form the bioactive composition in vitrified mixture solution 145 in container 170 .

[0123] like Figure 1A , the liquid in the vitrified mixture solution 145 is removed 150 in the system 155. A vacuum may be pulled on the container 170 containing the bioactive composition / vitrified mixture solution 145 to accelerate the removal of the liquid 115.

[0124] In some embodiments, the container 170 with the bioactive composition / vitrified mixture solution 145 can be heated to facilitate mixing of the components and formation of the vitrified bioactive composition. The mixture can be heated to between about 20 and 125 degrees Celsius.

[0125] In some embodiments, the container with the bioactive composition in the vitrified mixture solution 145 can be cooled. Specifically, the mixture with the bioactive composition / vitrified mixture solution 145 can be cooled to between 0 and -80 degrees Celsius. In embodiments, the mixture can be cooled until it is frozen. In the case of a frozen mixture, the frozen bioactive composition in the vitrified mixture solution 145 can be vacuumed to remove the liquid that was frozen with the bioactive composition in the vitrified mixture solution 145. After removing the liquid that was frozen with the bioactive composition in the vitrified mixture solution 145, the remaining powder will be formed from the vitrified bioactive composition.

[0126] FIG. 1B is a schematic diagram of an embodiment 175 of the structure of a vitrified bioactive composition 180 according to an embodiment. The formation of the vitrified bioactive composition 180 is a complex involving IDPs 120, sugars 130, and bioactive composition 110. The detailed formation mechanism of carbohydrate-driven cell protection includes the formation of a glassy matrix by the sugars within the cell, which physically prevents protein denaturation, protein aggregation, and membrane fusion of the IDPs 120. When the cells are dried, the water in the hydrogen bonds between the water in the solution of the IDPs 120 and the sugars 130 and the cell components is replaced by trehalose, which will also prevent protein denaturation of the IDPs 120 and aggregation and membrane fusion of the sugars 130. That is, even in the presence of trehalose, the replacement of hydrogen bonds between IDPs (e.g., silk fibroin) molecules and water by intermolecular and intramolecular hydrogen bonds can also be thermodynamically favorable, and the protein can undergo structural changes. Although trehalose induces protein vitrification, reconstruction, water loss, and volume changes still occur. This vitrified bioactive composition can also be referred to as an IDP / sugar / bioactive complex. Alcohol can also be used as a solvent. In particular, ethanol can be used as a liquid to form a vitrified mixture solution.

[0127] The formation of a glassy matrix within the vitrified IDP / sugar / bioactive complex preserves the bioactive composition. Surprisingly, particles and / or powders of the bioactive ingredient can be preserved by mixing with and into the vitrified IDP / sugar, thereby protecting the bioactive composition from deterioration. Specifically, both the process of forming the vitrified bioactive composition and the vitrified bioactive composition itself can preserve and protect the bioactive composition.

[0128] Once formed, further, the vitrified IDP / sugar / bioactive matrix (e.g., vitrified bioactive composition) can be processed to prepare a vitrified bioactive composition in the form of a powder, a pill, a tablet, a bead, etc. The processed vitrified bioactive composition can be ground into a powder. In some embodiments, the powder is in a particle size range of about 500 nanometers (e.g., nm) to about 1 millimeter (e.g., mm). In some embodiments, the powder is in a particle size range of about 1 micron to about 500 microns. In some embodiments, the powder is in a particle size range of about 5 microns to about 50 microns.

[0129] The powder of the vitrified bioactive composition, whether used directly or after grinding, can be processed into pellets. In some embodiments, the pellets can have a size between about 0.2 mm and about 2.0 mm. The pellets can be made into tablets and capsules. They can also be incorporated into polymer capsules.

[0130] Incorporating the bioactive ingredient into a glassy matrix essentially disperses the powder / particles in a glassy matrix, thereby preserving the powder / particles in a form that can be applied externally to humans, animals and plants, and can be delivered to humans or animals orally. In addition, the resulting vitrified bioactive composition is prepared in a measurable dose so that it can be packaged and administered in a calibrated dose.

[0131] The formation of a glassy IDP / sugar / bioactive matrix (e.g., a vitrified bioactive composition) incorporating powders / granules of one or more bioactive ingredients into a glassy matrix is ​​different from simply coating a larger item (e.g., a vegetable). While coating vegetables (e.g., avocados) with an IDP, sugar, or combination of IDPs and sugars can reduce the spoilage of the vegetable, coating the vegetable is different from preserving one or more powdered bioactive ingredients in a glassy matrix. For example, spraying or dipping the vegetable or fruit is different from dispersing the powder in a vitrification solution, preparing a vitrified bioactive composition powder for direct application or placing it in a cream that can be applied externally to humans, animals, or plants, or delivered orally to humans and animals. In addition, the vitrified bioactive composition prepared can be dosed in a measurable manner so that it can be packaged and administered in a calibrated dose.

[0132] 1C is a schematic diagram of a method for preparing an enteric encapsulated vitrified bioactive composition according to an embodiment. A certain amount of vitrified bioactive composition 190 is provided for encapsulation. The vitrified bioactive composition 190 can be in the form of a powder, pellets, or beads.

[0133] The vitrified bioactive composition 190 can be packaged in a capsule 192, which can be made of an enteric material or can be coated with an enteric material. In some embodiments, the enteric material is a polymer. The enteric polymer can also be a coating on the capsule 192. In this embodiment, the vitrified bioactive composition is encapsulated in a capsule having an enteric polymer coating. In embodiments, the vitrified bioactive composition can also be encapsulated in a capsule made entirely of an enteric polymer.

[0134] Enteric-encapsulated vitrified bioactive composition 194 can be administered as an oral drug. Enteric encapsulation prevents the vitrified bioactive composition from dissolving or decomposing in the gastric environment. That is, the enteric coating can prevent the polymer-coated oral drug from dissolving or decomposing in the stomach, so that the drug can be delivered to the lower gastric digestive tract of the subject. Some bioactive ingredients are unstable at the pH of gastric acid (pH about 2) and need to be prevented from degradation. Enteric coating is also an effective method for bioactive targeted therapy of bioactive compositions for the intestinal microbiome.

[0135] Enteric encapsulated vitrified bioactive compositions are not limited to being encapsulated in capsules. In some embodiments, vitrified bioactive compositions can be prepared as tablets, pills or beads coated with enteric polymers. Tablets, mini-tablets, pills and granules are the most common enteric-coated dosage forms. Enteric polymers can be applied to the surface of tablets, mini-tablets, pills and powders. Each tablet, mini-tablet, pill and powder is coated with an enteric polymer coating, and its structure allows each particle in the powder, pill, mini-tablet and tablet to be completely covered. That is, regardless of the size of the specific bioactive ingredient, the coating used can be an enteric polymer coating.

[0136] FIG2 is a schematic diagram of some potential embodiments of applications of vitrified bioactive compositions according to embodiments. Vitrified bioactive compositions 205 can be prepared and packaged for a variety of purposes. In some embodiments, vitrified bioactive compositions 205 can be packaged as oral therapeutics to improve intestinal health, or formulated in medicines to improve skin health 210.

[0137] In some embodiments, the vitrified bioactive composition 205 can be packaged as an oral medication to provide treatment 220. Specifically, as described above, the vitrified bioactive composition 205 can be packaged as enteric-coated capsules, tablets, and / or pills, which can be targeted for delivery to achieve treatment 220 based on the type of enteric material incorporated therein, according to the dissolving coating to different areas of the digestive system. In addition, the vitrified bioactive composition 205 can include enzymes and / or antibiotics 240 to be delivered to the biological subject.

[0138] In some embodiments, the vitrified bioactive composition 205 can be used in food processing. For example, the vitrified bioactive composition 205 can be used to aid in the fermentation 250 of food.

[0139] In some embodiments, the vitrified bioactive composition 205 can be used to stimulate plant growth 230. In some embodiments, the vitrified bioactive composition 205 can be delivered to plants, seeds, and / or soil by applying the powder and / or pellets of the vitrified bioactive composition by spraying a solution containing the enteric-encapsulated particles and / or powder of the vitrified bioactive composition. The bioactive composition 205 on the plant, seed, or soil.

[0140] In some embodiments, the vitrified bioactive composition 205 can be enteric-encapsulated to protect the bioactive composition from spoilage, from being eaten by insects, from being exposed to environmental conditions, and the like.

[0141] 3 is a schematic diagram of an embodiment of a method 300 for delivering a bioactive composition according to an embodiment. A bioactive composition may be provided in the method 300. The bioactive composition may include at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin, or an mRNA.

[0142] The bioactive composition is stored at step 320 to produce a vitrified bioactive composition. The storage of the bioactive composition includes vitrifying the bioactive composition in a vitrified mixture of intrinsically disordered proteins and sugars to produce a vitrified bioactive composition. The composition can release the bioactive composition from the vitrified mixture.

[0143] The vitrified bioactive composition can be administered to a biological subject in step 330. The administering includes applying it to a microbiome of the biological subject.

[0144] 4 is a schematic diagram of an embodiment of a method 400 for delivering a bioactive composition according to an embodiment. Step 410 provides a bioactive composition. The bioactive composition includes a bioactive substance, such as at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin, or an mRNA.

[0145] The bioactive composition may be stored to produce a vitrified bioactive composition in step 420. Storing the bioactive composition includes vitrifying the vitrified mixture of intrinsically disordered proteins and carbohydrates to produce a vitrified bioactive composition. The composition may release the bioactive composition from the vitrified mixture.

[0146] An enteric encapsulation is provided at step 430. In some embodiments, the enteric encapsulation can be a capsule having an enteric polymer coating. In some embodiments, the enteric encapsulation can be a coating applied to the vitrified bioactive composition.

[0147] The vitrified bioactive composition is encapsulated in an enteric package at step 440. In some embodiments, the enteric package encapsulates the vitrified bioactive composition in a capsule comprising an enteric polymer. Alternatively, in some embodiments, the enteric package coats particles of the vitrified bioactive composition with an enteric material.

[0148] The enteric-encapsulated vitrified bioactive composition is administered to a biological subject at step 450. The administration includes applying the enteric-encapsulated vitrified bioactive composition to a microbiome of the biological subject.

[0149] 5A is a schematic diagram of a method for preserving a bioactive composition according to an embodiment. In step 510, a vitrified solution may be formed by adding an intrinsically disordered protein and a sugar to a liquid to provide the vitrified solution, and then mixing the liquid.

[0150] A bioactive composition is added to the vitrification solution at step 520. The bioactive composition includes a bioactive agent of at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin, or an mRNA.

[0151] The bioactive composition is mixed in the vitrification solution at step 530. The mixing process can be accomplished by a mechanical mixer, a magnetic mixer, manual mixing, or the like.

[0152] Liquid is removed from the bioactive composition in the vitrification solution at step 540. The liquid removal process may be performed by at least one of air drying, freeze drying, vacuum drying, or heat drying.

[0153] FIG. 5B is a schematic diagram of a method of preparing a vitrification solution according to an embodiment.

[0154] A liquid is provided at step 560. The liquid may be water or an alcohol. In particular, the alcohol may be ethanol.

[0155] An intrinsically disordered protein is added to the liquid at step 570. The intrinsically disordered protein may be a naturally occurring protein, or a synthetic polymer.

[0156] Sugar is added to the liquid at step 580. The sugar may include one or more of a sugar, a disaccharide, or a monosaccharide.

[0157] In step 590, intrinsically disordered proteins and carbohydrates are mixed in a liquid to form a vitrified solution.

[0158] The following examples are intended to further illustrate the present invention and its preferred embodiments.

[0159] Example

[0160] Example 1

[0161] Example 1 represents an example of applying the bioactive microbial composition directly to the outside of the tablet as a film coating, which is accomplished by spraying the tablets with the microorganisms mixed into the IDP / disaccharide mixture. In this example, the premixed microorganisms and IDP / disaccharide mixture are sprayed directly into a gently rotating tablet drum. After thorough coating, the tablets continue to rotate in the drum until completely dry. At this point, the vitrified bioactive microbial coated tablets are ready for use.

[0162] The tablet itself can contain any therapeutically effective load component, such as vitamins, microbial prebiotics, small molecules for disease treatment, biologics, etc. Such vitrified bioactive microbial coated tablets are most effective for delivering the effective load component and encapsulated microorganisms to the intestines via oral administration. After the vitrified bioactive microorganisms reach the intestines, the biodegradable IDP / disaccharide mixture material is dissolved and the microorganisms are deposited in the intestinal lumen, where they exert their desired therapeutic or probiotic effects.

[0163] Example 2

[0164] Example 2 represents an embodiment of forming a powdered vitrified bioactive composition. A mixture of the bioactive composition is added to a vitrified solution of silk fibroin, disaccharide and water to form a bioactive composition vitrified solution (e.g., a vitrified mixture solution). The vitrified mixture solution is first cooled until it is frozen at -80 degrees Celsius to form a frozen vitrified mixture, and then the frozen vitrified mixture is placed in a freeze dryer for about 48 hours. The freeze dryer removes the frozen vitrified mixture in the process and leaves a powder of the vitrified bioactive composition.

[0165] The powder can be applied directly to humans or animals. The powder can be applied to the skin, hair, penis, vagina or feet, external auditory canal, nostrils and / or oral cavity.

[0166] Alternatively, a powdered vitrified bioactive composition can be dispersed in a lotion or gel and then applied to the same areas as above.

[0167] In some embodiments, prebiotics and / or microorganisms may also be mixed with the bioactive composition prior to mixing into the vitrification solution.

[0168] Example 3

[0169] Example 3 represents an example in which the powdered vitrified bioactive composition is directly mixed with food or beverage and ingested. The ingested vitrified bioactive composition can reach the microbiome in the oral cavity, esophagus, intestines, colon, etc. In this example, the powdered vitrified bioactive composition does not have an enteric coating.

[0170] Example 4

[0171] Example 4 represents an example of an enteric coated powdered vitrified bioactive composition in tablet form. The enteric encapsulated vitrified bioactive composition is ingested, and the vitrified bioactive composition remains intact within the enteric encapsulation until it reaches the intestine. Once in the intestine, the enteric coating breaks down, allowing the vitrified bioactive composition to be broken down or dissolved by mucus and other fluids present in the intestine, thereby delivering the bioactive composition to the intestinal microbiome, as well as microbial populations in the oral cavity, esophagus, intestines, and colon, etc.

[0172] Example 5

[0173] Example 5 represents an example in which a powdered vitrified bioactive composition is mixed with a filler (and optionally a prebiotic) and compressed into a tablet form. In this example, the powdered vitrified bioactive composition compressed into a tablet with a filler does not have an enteric coating. However, the filler and vitrified composition together can provide protection for the bioactive composition from acidic stomach acid and alkaline bile, as well as proteases in the intestine. In this example, the tablet of the vitrified bioactive composition mixed with a filler is able to reach the lower gastrointestinal tract, affecting the microbiome there.

[0174] Example 6

[0175] Example 6 represents an embodiment in which microorganisms are retained in a liquid or gel solution of IDP and disaccharide. In this embodiment, the gel solution is made of a gel-forming material, such as gelatin (made of collagen), and loaded with prebiotics to support microbial growth. The microorganisms in the liquid or gel vitrified solution are loaded into enteric-coated capsules (e.g., pH-activated capsules), which will rupture once the capsules pass through gastric acid and bile, releasing a mucus gel loaded with encapsulated commensal microorganisms.

[0176] Example 7

[0177] Example 7 represents an embodiment of enhancing the health of human skin microbiota and controlling the release of microorganisms and other bioactive ingredients in a sustainable and effective manner. In addition to the areas mentioned above, the embodiments can be applied to the following areas: skin, nostrils, mouth, vagina, hair, feet, and penis.

[0178] In some embodiments, after freeze-drying the vitreous bioactive composition mixture, it can be applied to the biological subject in powder form or by adding water or polymer to reconstitute the solution. The film can be applied to the face mask or swallowed, and the gel can be applied to the skin or attached to the mouth. The solution can be applied to the skin, mouth or nose as a spray.

[0179] In some embodiments, a mixture of intrinsic disordered proteins (IDPs) and disaccharides can be mixed with live or inactivated microorganisms alone or together. The mixture can be enhanced by adding some metabolites or preservatives. The mixture is air-dried into a film, which can be water annealed or ethanol annealed to promote the solidification of silk protein and sugar in the composite. The film can then be ingested to deliver the microorganisms to the oral cavity. The film can also be delivered through a facial mask and added to a band-aid to deal with injuries.

[0180] In some embodiments, a solution containing a vitrified bioactive composition comprising a microorganism and other bioactive ingredients may be formed. The solution may have preservatives or metabolites added to enhance performance. The solution may be applied as a spray to the skin, hair, nostrils, vagina, feet, and / or penis of a subject. The solution should be stored in a sealed container. The solution may also be used as a nasal spray for application to the nostrils, esophagus, and lungs.

[0181] The above embodiments of the present invention are exemplary only; many changes and modifications are obvious to those skilled in the art. These changes and modifications should fall within the scope of the present invention as defined by any one of the appended claims.

Claims

1. A method for delivering a biologically active composition to a biological subject, characterized in that: include: (1) providing a bioactive composition, wherein the bioactive composition comprises at least one bioactive substance selected from the group consisting of microorganisms, cells, tissues, vaccines, probiotics, antibiotics, vitamins, peptides, RNAi or mRNA; (2) preserving a bioactive composition, wherein the bioactive composition in a vitrified mixture composed of intrinsically disordered proteins and carbohydrates is vitrified to produce a vitrified bioactive composition, wherein the vitrified bioactive composition can release the bioactive composition from the vitrified mixture; (3) administering the vitrified bioactive composition to a biological subject, including administering the vitrified bioactive composition to a microbiome of the biological subject.

2. The method of delivering a biologically active composition to a biological subject according to claim 1, further comprising: Provided is an enteric encapsulation of a vitrified bioactive composition, characterized in that: The enteric encapsulation is used to protect the vitrified bioactive composition from the first set of environmental conditions; at the same time, the enteric encapsulation is used to decompose under the second set of environmental conditions to release the vitrified bioactive composition.

3. The method of delivering a biologically active composition to a biological subject according to claim 2, characterized in that: The enteric encapsulation is: placing the vitrified bioactive composition in an enteric polymer capsule or encapsulating the vitrified bioactive composition with an enteric coating.

4. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The intrinsic disordered protein comprises at least one of tardigrade intrinsic disordered protein, silk fibroin or tropoelastin; and the carbohydrate is a disaccharide.

5. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The microorganism comprises at least one of fungi, eubacteria, eukaryotes or archaea.

6. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The bioactive composition is: anaerobic, aerobic, spore-forming, vegetative, a human microbiome, an animal microbiome, a non-human microbiome, a non-animal microbiome, a plant microbiome, a non-plant microbiome, an artificial version of a bioactive composition of a non-human microbiome, or an artificial version of a bioactive composition of a non-animal microbiome.

7. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The activity of the bioactive composition is enhanced by adding metabolites and / or preservatives.

8. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The biological subject is a human being.

9. The method of delivering a biologically active composition to a biological subject according to claim 8, characterized in that: The vitrified bioactive composition is applied to at least one of the skin, vagina, penis, foot or armpit of a human.

10. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The administration method comprises ingesting the enteric-coated encapsulated vitrified bioactive composition, wherein the ingestion form of the vitrified bioactive composition is at least one of an oral pill, an oral tablet or a solution.

11. The method of claim 10 for delivering a biologically active composition to a biological subject, wherein: The enteric-coated vitrified bioactive composition is ingested in the form of an enteric-coated vitrified bioactive composition that enters the digestive tract of an organism; the enteric-coated vitrified bioactive composition passes through the digestive tract intact until it passes through an environment with a pH greater than 6, at which time the enteric-coated vitrified bioactive composition decomposes and releases the vitrified bioactive composition; thereafter, the vitrified bioactive composition releases the bioactive composition.

12. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The vitrified bioactive composition can reduce the metabolism of the bioactive substances in the bioactive composition, thereby placing the bioactive substances in a stagnant state.

13. The method of claim 11 for delivering a biologically active composition to a biological subject, wherein: Vitrifying the bioactive composition can prevent the bioactive composition from dying due to exposure to oxygen.

14. The method of claim 10 for delivering a biologically active composition to a biological subject, wherein: When the enteric-coated encapsulated vitrified bioactive composition passes through an environment with a pH greater than 6, the vitrified bioactive composition is released from the enteric-coated encapsulated vitrified bioactive composition in the intestinal tract of the biological subject; The vitrified bioactive composition is not released from the enterically encapsulated vitrified bioactive composition in the stomach of a biological subject.

15. The method of claim 14 for delivering a biologically active composition to a biological subject, wherein: The vitrified bioactive composition released from the intestinal tract of the biological subject adheres to intestinal mucus, rehydrates, revives, and proliferates; the bioactive composition is released from the vitrified bioactive composition over a period of time.

16. The method of claim 15 for delivering a biologically active composition to a biological subject, wherein: The bioactive composition released in the intestinal tract of the biological subject is at least one of live and healthy microorganisms, cells, tissues, vaccines, probiotics, antibiotics or mRNA.

17. The method of claim 4 for delivering a biologically active composition to a biological subject, wherein: The silk fibroin is extracted from silkworms; and the disaccharide is trehalose.

18. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The probiotics are used in the bioactive composition to provide energy to nourish the bioactive substances.

19. The method of claim 1 for delivering a biologically active composition to a biological subject, wherein: The biological subject is a plant, a seed or soil; The administration method is to spray a solution containing the enteric-encapsulated vitrified bioactive composition granules and / or powder onto plants, seeds or soil.

20. A composition for use in a microbiome of a biological subject, characterized in that: include: A bioactive composition and a vitrified mixture, wherein the bioactive composition includes at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin or an mRNA; the vitrified mixture is composed of intrinsically disordered proteins and carbohydrates, and the bioactive composition is preserved in the vitrified mixture to form a vitrified bioactive composition.

21. The composition according to claim 20, characterized in that: The composition further comprises: An enteric encapsulating agent is used to encapsulate the vitrified bioactive composition, protect the vitrified bioactive composition from a first set of environmental conditions, and release the vitrified bioactive composition under a second set of environmental conditions.

22. The composition according to claim 20, characterized in that: The bioactive composition is freeze-dried and stored in a vitrified mixture.

23. The composition according to claim 20, characterized in that: The bioactive composition may also include probiotics and / or antibiotics.

24. The composition according to claim 20, characterized in that: The vitrified bioactive composition is incorporated into a skin care solution.

25. The composition according to claim 20, characterized in that: The vitrified bioactive composition is incorporated into a wound healing medicament.

26. The composition according to claim 20, characterized in that: The intrinsically disordered protein is silk fibroin; and the carbohydrate is a disaccharide.

27. The composition according to claim 20, characterized in that: The intrinsically disordered protein is a naturally occurring protein or a synthetic polymer.

28. A method for delivering a biologically active composition to a biological subject, characterized in that: include: Providing a bioactive composition, the bioactive composition comprising at least one bioactive substance; storing a bioactive composition, the storing comprising vitrifying the bioactive composition in a vitrified mixture consisting of intrinsically disordered proteins and carbohydrates to produce a vitrified bioactive composition, the vitrified bioactive composition being used to release the bioactive composition from the vitrified mixture; Enteric-coated packaging available; encapsulating the vitrified bioactive composition in an enteric-coated capsule; The enteric-encapsulated vitrified bioactive composition is administered to a biological subject, the administration comprising administering the enteric-encapsulated vitrified bioactive composition to a microbiome of the biological subject.

29. The method according to claim 28, characterized in that: The enteric-coated package comprises: encapsulating the vitrified bioactive composition in a capsule containing an enteric polymer; or An enteric coating material is used to cover the particles of the vitrified bioactive composition.

30. The method according to claim 28, characterized in that: The bioactive agent comprises at least one of a microorganism, a cell, a tissue, a vaccine, a probiotic, an antibiotic, a vitamin or an mRNA.

31. The method according to claim 28, characterized in that: The intrinsically disordered protein is silk fibroin; and the carbohydrate is a disaccharide.

32. A method for preserving a biologically active composition, characterized in that: include: Provide fluids, Adding intrinsically disordered proteins to the liquid; Add sugars to the liquid; Mixing intrinsically disordered proteins with sugars in a liquid to form a vitrified solution; adding a bioactive composition to the vitrification solution; mixing the bioactive composition in the vitrification solution; as well as Liquid is removed from the bioactive composition in the vitrification solution.

33. The method according to claim 32, characterized in that: Also includes: The bioactive composition in the vitrification solution is heated to 20°C to 125°C.

34. The method according to claim 32, characterized in that: Also includes: The bioactive composition in the vitrification solution is cooled to 0°C to -80°C.

35. The method according to claim 32, characterized in that: The method of removing the liquid includes at least one of air drying, freeze drying, vacuum drying or heat drying.

36. The method according to claim 32, characterized in that: The liquid is water or alcohol.

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